PROJECT SUMMARY Many diseases in complex, hierarchically organized tissues such as the breast, lung, and prostate have been difficult to address, because they are a product of complex multicellular dynamics. For example, congenital diseases of the kidney are staggeringly common. Around a third of all birth defects are associated with problems in kidney and urinary tract development, but researchers have few options for capturing the full functional complexity of this organ system outside of the body. This is because current kidney models are either 2D, single cell-type approximations, or are organoid models with more cellular diversity, but with little of the long-range spatial structure that is crucial for kidney function. The Hughes lab aims to solve two critical engineering barriers to the development of better in vitro human tissue models. First, we aim to standardize and vastly increase the throughput of organoid- based phenotypic screens related to human disease. Second, we aim to bring an entirely new philosophy to tissue engineering, in which tissue scaffolds are not built in final form, but rather as immature ?seeds? that are guided through developmental transitions in structure that mimic those of their target tissue. These transitions morph flat tissue scaffolds into final tissue forms that achieve defined shapes, cell distributions, and ECM compaction and alignment patterns in 3D that establish a new way of building hierarchical tissues like the kidney. To the first aim, we propose to re-engineer our cell DNA ?velcro? cell and organoid patterning technology. This technology allows us to precisely pattern multiple cell types with single-cell resolution at the interface with organotypic gel layers, yet its throughput is currently limited. We will apply a photopatterning approach in which cell-adhesive ssDNA strands can be patterned in millions of locations simultaneously, a key requisite for whole-genome organoid screens. Secondly, we propose high-throughput pluripotent stem cell patterning and culture technologies that reduce inter-organoid variation, to enable whole genome CRISPR- based screening for genetic risk factors of disease, using kidney organoids as a prototypical system. To the second aim, we build upon our recent description of dynamic tissue scaffolds to position organoids in 3D using autonomously folding gels that couple their niches through tracts of dynamically remodeled ECM. Using these centimeter-scale, 3D organoid patterning capabilities, we envision an analogy between the branching pattern of the kidney collecting duct network and the edge networks of ?flasher? origami patterns. By controlling the morphogenesis of these patterns, we seek to engineer the progressive formation of a contiguous collecting duct network between locally self-organizing tissue niches. Rather than directly building tissues in a final, yet immature form, we believe that building hierarchical tissues by guided morphogenesis presents a transformative opportunity for modeling tissue homeostasis and disease.